Download Genetic risk factors for chronic obstructive pulmonary disease

Copyright ERS Journals Ltd 1997
European Respiratory Journal
ISSN 0903 - 1936
Eur Respir J 1997; 10: 1380–1391
DOI: 10.1183/09031936.97.10061380
Printed in UK - all rights reserved
REVIEW
Genetic risk factors for
chronic obstructive pulmonary disease
A.J. Sandford, T.D. Weir, P.D. Paré
Genetic risk factors for chronic obstructive pulmonary disease. A.J. Sandford, T.D.
Weir, P.D. Paré. ERS Journals Ltd 1997.
ABSTRACT: Cigarette smoking is the major risk factor for chronic obstructive
pulmonary disease (COPD). However, only a minority of cigarette smokers develop
symptomatic disease. Studies of families and twins suggest that genetic factors also
contribute to the development of COPD. We present a detailed literature review
of the genes which have been investigated as potential risk factors for this disease.
The only established genetic risk factor for COPD is homozygosity for the Z
allele of the α1-antitrypsin gene. Heterozygotes for the Z allele may also be at increased risk. Other mutations affecting the structure of α1-antitrypsin or the regulation of gene expression have been identified as risk factors.
Genes, including those for α1-antichymotrypsin, α2-macroglobulin, vitamin Dbinding protein and blood group antigens, have also been associated with the development of COPD. Variants of the cystic fibrosis transmembrane regulator gene
have been identified as risk factors for disseminated bronchiectasis.
The genetic basis to chronic obstructive pulmonary disease has begun to be elucidated and it is likely that several genes will be implicated in the pathogenesis of
this disease. The knowledge gained from such studies may also prove relevant to
other inflammatory diseases.
Eur Respir J 1997; 10: 1380–1391.
Chronic obstructive pulmonary disease (COPD) is characterized by decreased expiratory flow rates, increased
pulmonary resistance and hyperinflation. The most important risk factor for the development of COPD is cigarette smoking [1]. Cigarette smoke, in combination with
other factors, leads to two pathophysiological processes in the lung. The first is proteolytic destruction of the
lung parenchyma, which increases the size of the airspaces; these eventually coalesce to form emphysematous
spaces. The development of emphysema is associated
with a loss of lung elastic recoil. The second process is
inflammatory narrowing of peripheral airways, which is
characterized by oedema, mucus hypersecretion and
fibrosis, scarring, distortion and obliteration of peripheral airways. The loss of lung elastic recoil and the narrowing of the peripheral airways combine to decrease
maximal expiratory flow from the lung and contribute
to hyperinflation. In conjunction with gas exchange abnormalities, hyperinflation produces the symptoms of
COPD.
Despite the clear association of smoking and airway
obstruction, there remains marked interindividual variation in the response to cigarette smoke. This indicates
that there are additional genetic or environmental cofactors, which contribute to the development of COPD.
It has been estimated that only 10–20% of chronic heavy
smokers will ever develop symptomatic COPD [2, 3].
Co-factors, such as childhood viral respiratory infections
and environmental and occupational pollution, undoubtedly play a role in determining this susceptible subset.
Furthermore, there is evidence that genetic susceptibility
Respiratory Network of Centres of Excellence, UBC Pulmonary Research Laboratory, St. Paul's Hospital, Vancouver,
B.C., Canada.
Correspondence: P.D. Paré
UBC Pulmonary Research Laboratory
St. Paul's Hospital
1081 Burrard Street
Vancouver
B.C.
Canada
Keywords: Chronic obstructive pulmonary
disease
genetics
risk factors
Received: November 7 1996
Accepted for publication February 28 1997
is of major importance. The epidemiological and clinical data that demonstrate a hereditary contribution to
the development of COPD are summarized in table 1.
Although the results of several of these studies show
an aggregation of COPD in families, there is no clear
Mendelian pattern of inheritance. Case-control studies
have shown an increased prevalence of COPD in the
relatives of cases as compared to the relatives of controls, which cannot be explained by differences in other
known risk factors. There is also a higher prevalence
Table 1. – Studies that demonstrate a genetic component to the development of COPD
Study type
[Ref.]
Study showing clustering of COPD in
[4]
families
Family studies showing increased incidence
[5–12]
of COPD or chronic bronchitis in relatives of
cases compared to relatives of controls
Studies showing significant correlations in
[4, 7, 13, 14]
lung function between parents and children
and between siblings, and higher correlation
between parents and children, or between
siblings than between spouses
Studies showing decreased prevalence of
[5, 15, 16]
disease or less similarity in lung function
with increased genetic distance
Family studies showing a major gene effect
[17, 18]
or a genetic component to pulmonary function
Studies of pulmonary function in
[15, 19–24]
monozygotic and dizygotic twins
COPD: chronic obstructive pulmonary disease.
1381
GENETICS OF COPD
of reduced lung function among the children of patients
who have COPD than among their spouses. Cross-sectional studies have shown decreasing prevalence of disease and less similarity in lung function with increasing
genetic distance. Studies of twins support a large genetic contribution to the variability in lung function. Heritability estimates for forced expiratory volume in one
second (FEV1) range 0.5–0.8. WEBSTER et al. [21] studied the effects of smoking on lung function in monozygotic and dizygotic twins. They found that when one
monozygotic twin was susceptible to the effects of cigarette smoke, both twins developed reductions in lung
function, whereas other monozygotic twin pairs appeared to be nonsusceptible and, despite similar smoking
intensity, maintained normal lung function. The same
concordance of changes in the lung function with similar smoking intensity was not seen in dizygotic twins.
Figure 1 presents the pathogenic mechanisms in COPD
schematically.
Our purpose in this article is to review the evidence
that specific genes may contribute to genetic susceptibility to COPD.
Identification of susceptibility genes
Complex genetic diseases, such as COPD, are caused
by the interaction of environmental factors and genetic
susceptibility. Positional cloning has been used to identify the genes for many Mendelian disorders, and has
also proved successful in localizing multiple regions of
interest in complex diseases, such as hypertension [25]
and diabetes mellitus [26]. The positional cloning approach uses multiply-affected families, and compares the
inheritance of the disease to the inheritance of genetic
markers of known chromosomal location. If a genetic
marker is consistently co-inherited with the disease, then
it is inferred that the disease gene lies close to that marker on the same chromosome. Additional markers from
the region are used to progressively refine the localization, until the gene can be identified.
The power of positional cloning studies is reduced by
polygenic inheritance, genetic heterogeneity and interactions with environmental factors. Cigarette smoking
is such an important risk factor for COPD that it is
impossible to use family data in which the prevalence of
cigarette smoking varies. Ideally, one would need multigeneration families, in which there were similar levels
of exposure to cigarette smoke. However, this is extremely unlikely because of age- and gender-related differences in the prevalence of smoking. In addition, most
patients with COPD do not come to medical attention
until their fifth or sixth decade, by which time it is usually impossible to obtain phenotypic data and deoxyribonucleic acid (DNA) from their parents, and their
offspring are generally not old enough to have developed significant symptoms of COPD. An alternative approach would be to use an intermediate phenotype: a trait
which is known to predispose to the development of
COPD in smokers, such as increased bronchial responsiveness [27].
For these reasons, positional cloning is difficult to
apply to genes involved in the pathogenesis of COPD.
Therefore, an alternative strategy has been used; association studies of candidate genes. The candidate gene
approach involves identifying gene products that are
clearly involved in the pathogenesis of a condition, and
looking for genetic polymorphisms in the genes that
code for these proteins. To determine if these variants
contribute to the disease process, case-control studies are
performed to test for the association of the polymorphisms with the disease phenotype. The risk imparted by
a particular phenotype can be calculated using the relative risk (RR) or odds ratio (OR) equations. RR is given
by: (a/[a+b])/(c/[c+d]); and the OR is: (a/b)/(c/d), where
a and b are the number of patients with and without the
risk allele, respectively, and c and d are the number of
controls with and without the risk allele, respectively.
The calculation of OR and RR yields very similar values when the prevalence of a condition is low; however, the results diverge as the prevalence increases.
This is illustrated in figure 2, in which the RR and OR
for a genotype are calculated for different prevalences
of the trait in the population. An increased OR or RR
for a disease in individuals of a specific genotype may
indicate that the genotype causes an abnormal gene product or gene regulation, which influences the disease
pathogenesis. Alternatively, it is possible that the gene
tested in the association study does not contribute to
the disease process, but is in association with the true
80
Genetic
susceptibility
⇓ Lung recoil
70
Childhood respiratory
infections
⇓ Expiratory flow
hyperinflation
Airway inflammation
and remodelling
Symptoms
Fig. 1. – Summary of the pathogenic mechanisms in chronic obstructive pulmonary disease (COPD). Exposure to cigarette smoke is the
major factor in the pathogenesis of COPD but interacts with other
risk factors, including genetic susceptibility, to produce airway obstruction by loss of elastic recoil and/or airway inflammation.
60
Odds ratio
Environmental
and occupational
pollution
Cigarette smoke
50
40
30
20
10
0
0
0.1
0.2
0.3 0.4 0.5
Prevalence of trait
0.6
0.7
0.8
Fig. 2. – Dependence of estimates of relative risk (RR) on the population prevalence of the trait under study. Values for RR and odds
ratio (OR) diverge as the prevalence of the trait increases. ——: RR=5;
- - -: RR=20.
1382
A . J . SANDFORD ET AL .
disease-causing mutation. This is
Pulmonary
because the disease-causing mutaand bronchial
capillaries
tion may have first occurred on a
chromosome containing the genotype being tested in the study. If the
two alleles are very close to each
other, then they will remain in assoVitamin
ciation with each other for several D-binding protein,
Alpha1-antiprotease
chemotaxic
generations and are said to be in
CFTR
Alpha1-antichymotrypsin
with
C5a
linkage disequilibrium.
Alpha2-macroglobulin
The power of association studies
has been clearly demonstrated [28].
SLPI
Even genetic polymorphisms which
Cytochrome P450
impart only a slight increase in RR
Elastase,
can be detected if sufficient numbers
Bronchial
other proteases
Clara
hyperresponsiveness
of patients and controls are obtainand TNF-α
cell
genes
Interstitial
ed. The weakness of the candidate
SOD
gene approach is that only genes
known to be involved in the patho- Fig. 3. – Schematic representation of an airway to illustrate how mutations in various genes may
contribute to the development of chronic obstructive pulmonary disease (COPD). Alpha1-antiprotegenic process can be examined. The ase
(α1-antitrypsin), α1-antichymotrypsin, and α2-macroglobulin are serum proteins that can inhibit
other major difficulty is ensuring that inflammatory
cell proteases. Deficiencies in their function or level could enhance the proteolytic
the patient and control groups are digestion of the lung parenchyma that characterizes emphysema. Cytochrome P450 is an enzyme
adequately matched for every other present in airway epithelial cells (primarily Clara cells) that converts inhaled toxic chemicals to
variable that could influence the their metabolites. A gene variant, which enhances the enzyme's activity, could increase the prevalence of lung cancer as well as accelerate the airway inflammation that characterizes COPD. There is
distribution of the genotype. Chief an association between mutations in the CFTR gene and bronchiectasis. Variants of the vitamin Damong these is ethnic origin. There binding protein may influence the susceptibility to COPD. This protein can be converted to a mais potential for false-positive or crophage-activating factor and interact with complement factor 5a (C5a) and C5a des-Arg to enhance
false-negative results if this factor chemotaxis of inflammatory cells. CFTR: cystic fibrosis transmembrane regulator; SLPI: secretois not carefully taken into account. ry leucocyte proteinase inhibitor; TNF-α: tumour necrosis factor-α; SOD: superoxide dismutase.
indicates those genes for which association studies have
For instance, an association of type 2 diabetes mellitus
shown a significant relationship between specific polyand an immunoglobulin G (IgG) haplotype was shown
morphisms and COPD, and candidate genes that have
to be due to Caucasian admixture in a Native American
the potential to be involved in the pathogenesis of COPD
population [29]. Caucasians have a lower incidence of
but for which there are no significant associations at
diabetes and coincidentally a higher prevalence of the
present. Figure 3 is a schematic illustration of an airIgG haplotype. Therefore, the haplotype appeared to be
way to depict how enhanced or deficient gene products
protective against diabetes, but in fact was only a marcould contribute to COPD.
ker for Caucasian ancestry. The association was shown
to be spurious because the protective effect was not seen
in individuals with no Caucasian ancestry.
Alpha1-antitrypsin
Genetic factors in the pathogenesis of COPD
The recognition by LAURELL and ERIKSSON [30] that
Table 2 lists genes that have been tested as candidates
patients with extremely low levels of α-globulin had an
for involvement in the pathogenesis of COPD. The table
increased prevalence of emphysema was the first study
to show a genetic risk for COPD. Alpha1-antitrypsin (α1Table 2. – Genes implicated in the pathogenesis of
AT)
is a powerful antiprotease and is one of the few
COPD
enzymes that can inhibit leucocyte elastase. Alpha1-AT
Genes for which association studies have shown a signifiis produced in large amounts by the liver, but is also
cant relationship between polymorphisms and COPD
produced by alveolar macrophages and peripheral blood
Alpha1-antitrypsin
monocytes [31]. It is a highly polymorphic protein and
Alpha1-antichymotrypsin
over 70 variants have so far been identified [32] using
Cystic fibrosis transmembrane regulator
crossed electrophoresis [33] and isoelectric focusing [34].
Vitamin D-binding protein
The Z variant of α1-AT has deficient antiproteolytic funcAlpha2-macroglobulin
Cytochrome P450A1
tion but, more importantly, it is improperly processed
ABH secretor, Lewis and ABO blood groups
in the rough endoplasmic reticulum and aggregates withHLA
in the cell. Large amounts of the Z variant of the α1Immunoglobulin deficiency
AT protein accumulate in hepatocytes, where they can
Haptoglobin
cause liver disease [35]. Individuals with homozygous
Candidate genes for which there are no significant assoZ mutations have extremely low levels of circulating
ciations at present
α1-AT (less than 15% of normal) and have a clearly
Extracellular superoxide dismutase
Secretory leucocyte proteinase inhibitor
accelerated rate of decline in lung function even in the
Cathepsin G
absence of smoking [36, 37]. However, it is predominantly among smokers who are homozygous that sympCOPD: chronic obstructive pulmonary disease; HLA: human
tomatic airflow obstruction develops at a younger age
leucocyte antigen.
GENETICS OF COPD
[38, 39]. Although there is a clear association of homozygosity for this gene variant and the development of
COPD, the homozygous state is rare in the population
(1 in 1,670 [40] to 1 in 5,097 [41] live births in Caucasian
populations) and, thus, can explain only a small percentage of the genetic susceptibility to cigarette smoke.
The discovery that homozygosity for the Z variant
leads to increased risk for COPD led to numerous studies in which an association of COPD and heterozygous
genotypes was sought. The approximate allele frequencies of the most common gene variants M, S and Z are
0.93, 0.05 and 0.02, respectively. Patients with the MM
genotype have the highest α1-AT levels and are defined
as normal. Patients who are heterozygous MS have mild
reductions in α1-AT levels to ~80% of normal, whereas
MZ heterozygotes have lower levels at ~60% of normal.
SZ compound heterozygotes are rare, but have even
lower levels at ~40% of normal.
Two types of studies have attempted to identify an
increased risk for COPD in the relatively common heterozygous MS and MZ genotypes. In case-control studies,
the prevalence of α1-AT genotypes in individuals with
the clinical features of COPD is compared to control
subjects without airflow obstruction, who are matched
as closely as possible for other potential predictors of
COPD. In general, the results of these case-control studies have shown the OR to be significantly increased. As
shown in table 3, the OR for COPD ranges 1.5–5.0. The
prevalence of the MZ variant in the case populations
ranges 3.9–14.2%, whilst in the controls it ranges 1.0–
5.3%.
Investigators have also assessed the risk of the MZ
genotype by studying lung function in the general population [49–56]. In these studies, a population sample is
phenotyped for α1-AT variants and the prevalence of
COPD in those with the MZ phenotype is compared
with the prevalence in those with the MM phenotype.
Many of these studies were based on small numbers of
individuals and had insufficient power to detect an effect
of the MZ or MS phenotype. However, even most of
1383
the larger studies showed no significant difference in
respiratory symptoms or pulmonary function in the MZ
individuals compared to MM subjects. In theory, population-based studies designed to examine the predictive
value of a genotype are superior to case-control methods because there is less chance of a systematic bias.
However, in COPD, where an environmental factor (i.e.
cigarette smoking) plays an important role, population
studies may have insufficient sensitivity to detect a factor which only increases risk slightly. For example, in a
collaborative study to assess risk of lung disease in MZ
phenotype subjects, 143 MZ individuals did not have
significantly lower lung function than 143 MM individuals drawn from a population study of over 10,000
people [56]. However, only 37% of the subjects were
current smokers, 35% had never smoked and 60% were
less than 54 yrs of age.
In contrast to these reports, the results of several population studies have demonstrated differences between
MZ and MM individuals. KLAYTON et al. [57] found an
increased prevalence of COPD in MZ heterozygotes who
had smoked, but found no difference in the incidence
of COPD between MM and MZ nonsmokers. COOPER et
al. [58] found significantly decreased lung function in
MZ individuals. However, both of these studies used
relatives in the MZ study population and, therefore, the
results may not be due to mutations in the α1-AT gene.
TATTERSALL et al. [59] found evidence for greater loss
of elastic recoil in MZ versus MM smokers, but estimates of airway function were similar in both groups. HALL
et al. [60] found that MZ heterozygotes had significantly
lower expiratory flow rates, even in the absence of smoking. MADISON et al. [10] found more rapid decline in
lung function in MZ individuals in a longitudinal study.
Similarly, the results of a 10 year longitudinal study of
28 MZ subjects demonstrated that deterioration in lung
function was significantly greater than in a matched MM
control group [61].
In addition to mutations that affect the basal serum
levels of α1-AT, several mutations have been described that affect function [62], but
Table 3. – Case-control studies of α1-antitrypsin deficiency genotypes and chro- these are relatively rare and can
nic obstructive pulmonary disease (COPD)
only explain a small percentage
First
[Ref.]
Subjects
Genotypes %
OR
of the susceptible subgroup that
develops COPD. Two separate
author
MZ
MS
ZZ
SS
SZ
for
groups have reported an associMZ
ation between a mutation in the
SHIGEOKA
[42]
306 COPD patients
3.9
4.0
3' region of the α1-AT gene and
196 controls
1.0
COPD [63, 64]. KALSHEKER et al.
526 COPD patients
5.9
6.5
0.9
3.4
5.0
BARTMANN [43]
[63] found that this mutation was
642 controls
1.2
6.5
0.3
0.2
[44]
114 emphysema or
4.9
5.7
6.6
2.6
COX
associated with chronic lung disbronchitis patients
ease. Heterozygosity for the mu721 controls
1.9
7.9
0
tation in a group of patients with
JANUS
[45]
190 emphysema
14.2
5.3
2.6
1.1
3.9
pulmonary emphysema (18%),
patients
and in a group of patients with
1,303 controls
3.9
7
0.1
0.3
bronchiectasis (19%), was signiLIEBERMAN [46]
965 COPD patients
7.7 10.1
1.9
0.3
0.2
3.3
ficantly higher than in normal
1,380 controls
2.5
8.0
0
0.1
0.4
controls (5%). However, the reaMITTMAN
[47]
350 COPD patients
10.0
6.3
3.4
0.9
0.9
3.8
son for the association of the
2,830 controls
2.9
4.1
0.1
0.1
0.1
[9]
114 COPD patients
7.9
4.4
2.6
0
1.5
KUEPPERS
mutation with COPD was unclear,
114 controls
5.3
7.0
0
0.9
since it was not associated with
[48]
107 COPD patients
9.3
5.6
1.9
4.6
BARNETT
α1-AT deficiency or any partic91 controls
2.2
5.5
0
ular α1-AT protein type. Subsequently, these authors studied a
OR: odds ratio.
1384
A . J . SANDFORD ET AL .
larger group of 140 patients with pulmonary emphysema and bronchiectasis and found that 20% were heterozygous for the mutation (p=0.0015) [65]. The association
has been independently confirmed by POLLER et al. [64]
in a group of 137 COPD patients. The mutation was
found in 15% of the patients and in only 5% of the
healthy controls. In addition, a family was identified in
which the mutation segregated with COPD, and, when
homozygous, the mutation was associated with the onset
of symptoms at a younger age.
The 3' mutation could be associated with COPD as a
result of linkage disequilibrium with the disease-causing
allele. The α1-antichymotrypsin gene has been mapped
to within 220 kb of the α1-AT locus [66], and the mutant
3' allele could be in disequilibrium with an α1-antichymotrypsin deficiency allele. Alternatively, KALSHEKER
and co-workers [65] have suggested that the 3' mutation may affect the regulation of α1-AT gene expression. Alpha1-AT is an acute phase protein and its serum
concentration increases two- to threefold during inflammation [67]. Presumably, the acute phase response has
evolved to attenuate the proteolytic destruction that
occurs at sites of acute tissue injury and, thus, prevents
excessive tissue destruction. A deficient acute phase increase in α1-AT levels following viral or bacterial respiratory infections could exaggerate the proteolytic tissue
destruction that accompanies the release of neutrophil
elastase and other enzymes. It is possible that the 3' mutation could affect the acute phase response leading to
reduced upregulation of α1-AT synthesis when inflammation is present. Alveolar and lung tissue macrophages
are both capable of producing α1-AT [31]. If the α1-AT
gene expression in tissue and alveolar macrophages is
also affected by the mutation, then a disturbance of the
proteolytic-antiproteolytic balance could develop within the microenvironment of the inflamed lung.
MORGAN et al. [68] sequenced the 3' region of the
α1-AT gene, and showed that the mutation occurs in a
region containing four consensus sequences for DNAbinding proteins, suggesting that it may affect a regulatory element. Gel shift analysis and deoxyribonuclease
(DNase) I footprinting experiments confirmed that all
four potential regulatory regions bound nuclear factors
[69]. However, the mutant sequence demonstrated poor
binding, especially in the region of the mutation.
To test for the functional significance of the mutation,
both the wild type and mutant 3' regions were cloned
into vectors, downstream of a reporter gene. These constructs were used to transfect three different cell lines.
In all of the cell types, the wild type sequence showed
a 50–100% increase in gene expression compared to a
control plasmid. Furthermore, the mutant sequence showed two- to fourfold less activity than the wild type.
The acute phase response is primarily mediated by
interleukin 6 [70]. Recently, it has been proposed that
transcription factors of the CCAAT box enhancer binding protein (C/EBP) family play an important role in
increasing acute-phase gene transcription [71]. The 3'
region of the α1-AT gene contains a C/EBP binding site.
Interestingly, the mutation in the 3' region appears to
influence the binding to neighbouring regions, including the C/EBP site and, therefore, may influence acute
phase gene expression.
An additional polymorphism in the 3' region of the
α1-AT gene has been shown to be associated with COPD
[72]. The polymorphism was found in 3 out of 70 COPD
patients but in none of 52 controls. The mutant allele
showed loss of more than one restriction site, suggesting the presence of a deletion. Homozygosity for this
mutation was associated with early onset COPD. This
polymorphism was also associated with normal α1-AT
levels.
Alpha1-antichymotrypsin
Alpha1-antichymotrypsin, like α1-antitrypsin, is a serine protease inhibitor and acute phase reactant. Alpha1antichymotrypsin (α1-ACT) is known to inhibit pancreatic
chymotrypsin, neutrophil cathepsin G, mast cell chymase and the production of neutrophil superoxide [73].
It is synthesized by hepatocytes and alveolar macrophages [74].
Alpha1-ACT deficiency has a prevalence of approximately 1% in the Swedish population. In cases where
hereditary deficiency has been shown, transmission follows an autosomal dominant inheritance pattern [75,
76]. No consistent clinical phenotype is associated with
α1-ACT deficiency, although an increased prevalence
has been reported in patients with childhood asthma [77]
and COPD [78, 79]. In two other studies, deficient patients had increased values of residual volume (RV) and
of the RV/total lung capacity (TLC) ratio [75, 76].
Two point mutations in the α1-ACT gene have been
associated with decreased α1-ACT serum concentrations
and COPD. POLLER and co-workers [78] described an
amino acid substitution, Pro227→Ala, which they found
in four of 100 unrelated COPD patients and none of 100
controls in a German population (p=0.04). All four patients with the mutant gene had serum α1-ACT concentrations approximately 60% of normal and α1-AT levels
within the normal range. However the prevalence of the
Pro227→Ala mutation may vary in different populations,
since it was not detected in 102 Russian COPD patients [80]. A second amino acid substitution, Leu55→Pro,
was reported by POLLER and co-workers [79] in three out
of 200 unrelated COPD patients and none of 100 controls. Mean α1-ACT serum levels in the heterozygotes
was 80% of normal, and the mutant protein had an
altered pattern on isoelectric focusing and defective function. One of the heterozygotes belonged to a family in
which three members were affected with severe early
onset COPD. The mutant allele segregated with COPD
in this three generation pedigree.
Cystic fibrosis transmembrane regulator
The cystic fibrosis transmembrane regulator (CFTR)
gene product forms a chloride channel at the apical surface of airway epithelial cells and is intricately involved
in the control of airway secretions. Homozygous deficiency or defective function of this protein results in
cystic fibrosis (CF), characterized by elevated sweat
chloride levels and early onset obstructive lung disease,
secondary to chronic bacterial infection and bronchiectasis. The prevalence of CF is 1 in 2,000 to 1 in 3,000,
with the carrier frequency estimated at 1 in 20 to 1 in
1385
GENETICS OF COPD
30 in populations of Northern European descent [81].
It has been hypothesized that this relatively high prevalence arose from a selective advantage of carrying a
CF allele. Resistance to pulmonary tuberculosis [82],
influenza [83], and cholera [84] have each been suggested as a selective advantage. In an animal model, mice
that were heterozygous for a mutant CFTR allele secreted 50% less intestinal fluid and chloride ion in response
to cholera toxin [85].
CF heterozygotes could have altered airway water and
ion regulation, altered mucociliary clearance and an increased susceptibility to challenges that are attenuated
by these mechanisms. In the 1960s, several groups investigated the hypothesis that CF heterozygotes may be predisposed to respiratory disease. Comparisons of parents
of CF patients versus controls (mean age 34–36 yrs) did
not reveal any significant differences in lung function
or history of asthma or chronic bronchitis [86–89]. However, obligate heterozygotes have been shown to have
increased bronchial reactivity to methacholine [90], and
increased incidence of wheeze accompanied by decreased FEV1 and forced mid-expiratory flow (FEF25–75)
[91].
More than 580 variants of the CFTR gene have been
described; the most common mutation, ∆F508, is found
on approximately 70% of all CF chromosomes [92].
Heterozygosity for the ∆F508 mutation was identified
in four of eight patients with disseminated bronchiectasis [93], and in five of 65 patients with bronchial hypersecretion [94]. In both studies, it is unclear whether the
∆F508 heterozygotes are predisposed to lung disease
or whether they have mild, previously undiagnosed CF
with unidentified CFTR mutations on their other chromosomes. In a study of patients with normal sweat
chloride levels, GERVAIS et al. [95] found the prevalence
of ∆F508 to be increased (four out of 47) in patients
with bronchiectasis and not increased (seven out of
161) in patients with chronic bronchitis. The ∆F508
mutation was not found in any of 21 Japanese patients
with diffuse panbronchiolitis, a disease with pathological and clinical characteristics similar to mild CF [96].
Recently, investigators have searched for associations
between respiratory disease and other CFTR variants,
in addition to ∆F508. ARTLICH et al. [97] examined 100
patients with chronic bronchitis for the more common
CFTR mutations (∆F508, R553X, G551D, G542D,
G542X, N1303K and 621+1G→T). The only mutation,
∆F508, was found in one patient who also had bronchiectasis, suggesting that none of these CFTR mutations predisposes to chronic bronchitis [97, 98]. PIGNATTI
and co-workers [99] performed detailed screening for
approximately 70 CFTR mutations. Although variants
were found in two of 12 patients with COPD without
bronchiectasis, and in two of 36 patients with nonobstructive pulmonary disease, the frequency of the
mutations was not significantly different from that expected. However, CFTR mutations were found in five of 16
patients with disseminated bronchiectasis and normal
sweat chloride levels (one each with mutations ∆F508,
R75Q, M1137V, 3667ins4, R1066C). In a subsequent
study, five of the same 16 patients were also found to
have the IVS8-5T variant (three of whom were previously negative for other CFTR mutations) [100]. The
IVS8-5T allele results in reduced CFTR gene expres-
sion. This variant was not found to be significantly increased in five of 33 COPD patients. Early work by the
same authors did not support the involvement of CFTR
in COPD by linkage analysis with a CF locus marker
[101].
In summary, heterozygosity for ∆F508 appears to predispose for disseminated bronchiectasis, but the involvement of CFTR in other obstructive pulmonary diseases
remains unproven. Studies of CFTR mutations in COPD
patients who have documented lifelong airway challenges, such as cigarette smoking, have not been performed.
Vitamin D-binding protein (group-specific component)
Vitamin D-binding protein (VDBP), also known as
group-specific component, is a 55 kDa protein secreted
by the liver, that is able to bind extracellular actin and
endotoxin in addition to vitamin D. VDBP enhances the
chemotactic activity of complement factor 5a (C5a) and
C5a des-Arg for neutrophils by one to two orders of
magnitude [102]. In addition, VDBP can act as a macrophage-activating factor [103]. Thus, besides its vitamin
D-binding function, VDBP can have important influences on the intensity of the inflammatory reaction.
Numerous isoforms of VDBP have been identified by
isoelectric focusing. Two common substitutions in exon
11 of the gene result in three possible isoforms, termed
1F, 1S and 2. Figure 4 shows a partial gene map of
VDBP and the substitutions responsible for protein isoforms. KUEPPERS et al. [9] found a decreased frequency
of the 2-2 genotype in COPD patients compared to controls. Subsequently, HORNE et al. [104] performed a casecontrol study, in which they found that the prevalence
of the 1F homozygote was significantly greater among
patients with COPD than among controls, yielding a RR
of 4.8. In addition, the genotypes that contained the 2
allele (2-1F, 2-1S and 2-2), had a protective effect. However, this association remains controversial, since it was
not replicated by KAUFFMANN et al. [105].
a)
Intron 10
Exon 11
G → T
Glu416 → Asp
Intron 11
C → A
Thr420 → Lys
b)
Isoform
Amino acid 416
Amino acid 420
Population
frequency
1S
1F
2
Glu
Asp
Asp
Thr
Thr
Lys
0.57
0.18
0.25
Fig. 4. – Polymorphisms in the vitamin D-binding protein gene. a)
Two point-mutations in exon 11 of the gene result in amino acid substitutions at positions 416 and 420 of the protein. b) Amino acids present at position 416 and 420 in the three isoforms of the vitamin
D-binding protein, and the frequencies of the isoforms in Caucasian
populations. G: guanine; T: thymine; C: cytosine; A: adenine; Glu:
glutamic acid; Asp: aspartic acid; Thr: threonine; Lys: lysine.
1386
A . J . SANDFORD ET AL .
No studies have so far examined the influence of these
genetic variants on the ability of the protein to act as a
chemotactic enhancer of C5a or as a macrophage-activating factor. However, the macrophage-activating factor
is formed from VDBP by modification of an oligosaccharide side chain. Less than 10% of the 2 isoform is
glycosylated and able to form macrophage-activating
factor [103], which is consistent with a protective effect
for the 2 allele.
Alpha2-macroglobulin
Alpha2-macroglobulin is a broad spectrum serum protease inhibitor. Normal serum levels of α2-macroglobulin
are higher in females and decrease with age. Synthesized
by hepatocytes, alveolar macrophages [106] and human
lung fibroblasts [107], α2-macroglobulin is thought to
have a protective role in the lung. The large size of α2macroglobulin (725 kDa) prevents significant transport
from blood to the lung interstitium or alveolar space, so
that serum levels do not necessarily reflect its concentration in the lung. However, increased permeability of
the vessel wall under inflammatory conditions could
allow α2-macroglobulin to enter the interstitial space
[108]. An increase in α2-macroglobulin levels can be
detected in the sputum of patients with acute chest infections [109]. Elevated α2-macroglobulin levels, up to
two times control, have been reported in the serum of
patients with α1-AT deficiency, irrespective of the presence or absence of COPD [110, 111]. Such an elevation is not seen in patients with emphysema unrelated
to α1-AT deficiency.
Alpha2-macroglobulin serum deficiency is rare and the
cause is unknown. Two case studies described hereditary
α2-macroglobulin deficiency with autosomal dominant
transmission [112, 113]. Although symptoms suggestive
of respiratory disease were not found in the deficient
individuals, it is possible that the subjects were not old
enough to develop COPD. Neither study included pulmonary function tests or smoking histories. Complete
lack of α2-macroglobulin has not been described and
may be incompatible with life.
The α2-macroglobulin gene, located on chromosome
12, has been cloned and sequenced [114]. Whilst restriction fragment length polymorphism (RFLP) variants of the α2-macroglobulin gene have been described,
only one variant has been reported to be associated with
chronic lung disease in a single patient [115]. The patient
had α2-macroglobulin serum levels 50% of normal and
chronic pulmonary disease since childhood progressing
to very severe COPD at the age of 42 yrs (smoking history not reported). DNA from this patient was digested
with 10 restriction enzymes and probed with an α2macroglobulin complementary deoxyribonucleic acid
(cDNA) probe. All 10 restriction enzymes showed an
alteration in the RFLP pattern suggesting a major alteration of the α2-macroglobulin gene. RFLP analysis with
nine of the 10 restriction enzymes failed to demonstrate
polymorphisms in 40 control and 39 COPD patients.
The same author sequenced two functional domains of
the α2-macroglobulin gene in 30 COPD patients and 30
control subjects [114]. A common amino acid substitution, Val1000→lle, was detected equally in both groups.
One COPD patient had an amino acid substitution,
Cys972→Tyr, which was predicted to interfere with α2macroglobulin function. The serum level of α2-macroglobulin in this patient was within the normal range.
Cytochrome P4501A1
Cytochrome P4501A1 is an enzyme that metabolizes
exogenous compounds to enable them to be excreted in
the urine or bile. It is found throughout the lung, and
may play a role in the activation of procarcinogens to
their carcinogenic forms. The enzyme is produced by
the CYP1A1 gene and mutations at this locus have been
associated with lung cancer [116].
In a recent study, the prevalence of a mutation in exon
7 of CYP1A1 was assessed in lung cancer and COPD
patients [117]. This mutation causes a substitution of
isoleucine to valine at residue 462, and results in a protein with almost twice the enzymatic activity of the
isoleucine protein. The high-activity allele was found to
be associated with susceptibility to centriacinar emphysema and lung cancer. The polymorphism was not linked to lung cancer in the absence of emphysema.
Blood group antigens
The association of COPD with the ABO, secretor and
Lewis genes has been the focus of several studies. The
ABO locus on chromosome 9 determines the activity of
a glycosyltransferase, which converts glycoprotein H into
the A or B antigens. An association between the ABO
locus and COPD was found by COHEN et al. [118]. The
results of this study suggested that impaired lung function was associated with type A blood group. This was
confirmed by the same authors in a 5 yr longitudinal
study, in which there was a greater decline in lung function in group A individuals compared with non-A subjects [119]. In direct contrast to these studies, KRYZANOWSKI
et al. [120] found that subjects with blood group A had
a smaller decline in lung function than individuals with
other blood groups. The results of several other studies
have failed to confirm any association of ABO alleles
and pulmonary function [23, 121, 122].
ABO antigens are present on virtually all cells of the
body. Approximately 80% of the population secretes
ABO antigens into saliva, plasma and respiratory tract
secretions. The ability to do this is determined by the
secretor locus on chromosome 19q and is a dominant
trait. It has been reported that nonsecretors have impaired lung function compared to secretors [123, 124]. This
suggests that the presence of ABO antigens in the secretions of the respiratory tract may have a protective effect
against lung impairment. This result was independently confirmed by KAUFFMANN et al. [105], who found significantly more nonsecretors of blood group O in subjects
with low FEV1 measurements (OR=15.6). Secretor status was shown to have a protective effect against decline in peak expiratory flow rates [123], but, in this
study, the effect was only observed in subjects over 40
yrs of age. These associations are controversial because
they have not been replicated in other populations [121,
122, 125].
1387
GENETICS OF COPD
The Lewis blood group has also been investigated as
a possible risk factor for airflow obstruction [126]. In
Caucasian populations, ~90% of individuals have the
dominant Le allele and produce Lewis a substance. In
individuals who are secretors, this is converted to Lewis
b substance, and therefore they have a and b substances
in their serum. Lewis-positive nonsecretors only have
a substance in their serum. HORNE et al. [126] found a
significant increase in airflow obstruction in Lewisnegative subjects, with a RR of 7.2. The authors suggest that it is the presence of b substance rather than
secretor status that protects against airflow obstruction.
Since the blood group systems interact at the protein
level, a recent study has considered all three gene loci
together [127]. Blood group O individuals who were either
Lewis-negative or nonsecretors, were found to have
impaired lung function and higher prevalence of wheezing and asthma. Individuals who were both Lewisnegative and nonsecretors, had very low lung function.
Lewis-positive secretors were found to have lower lung
function if they had blood group A, compared with group
O.
The reason for the association of ABO, Lewis and
secretor genes with COPD remains unclear, but it may
be due to the role of these systems in the adhesion of
infectious agents [128]. Recurrent respiratory infections,
especially in childhood, are known to be a risk factor
for COPD, and particular alleles of these blood groups
may increase an individual's susceptibility to infection.
of IgG2 and two had decreased levels of IgG3 [130].
All six of the IgG and IgA deficient subjects were found
to have abnormal lung function. In addition, a significant correlation of IgG2 levels and FEV1 values was
found by O'KEEFE et al. [131]. Selective IgA deficiency
has been found to segregate with COPD, in a large, three
generation pedigree [133].
Haptoglobin
The serum protein haptoglobin has several common
polymorphisms. The prevalence of these variants was
investigated in a population of subjects with low FEV1
values [105]. Overall, no significant difference in the frequency of haptoglobin variants was observed between individuals with low FEV1 values compared to those with
high values. Among those with non-O blood groups, a
possible protective affect of the Hp1S allele was detected. However, a similar association was not found in an
earlier study [9].
Other candidate genes for COPD
Extracellular superoxide dismutase
Associations between the human leucocyte antigen
(HLA) class I genes and COPD have been investigated
in a study of heavy smokers with high FEV1 values and
lifelong nonsmokers with low FEV1 values [105]. The
authors observed a significant decrease of the HLABw16 allele in those with low FEV1 values (OR=0.2),
and a significant increase of the HLA-B7 antigen in the
same group (OR=3.8).
HLA typing was also performed in a population of
Japanese patients with diffuse panbronchiolitis [129].
The results demonstrated an increased prevalence of
HLA-Bw54 in the patients compared to the control subjects (RR=13.3). This HLA type is only found in Japanese, Chinese and Korean individuals, and may explain
why diffuse panbronchiolitis has not been reported in
Caucasian or African populations.
It is not yet clear whether these associations are due
to variations in the HLA genes themselves or to susceptibility genes in linkage disequilibrium with the HLA
alleles.
Extracellular superoxide dismutase (EC-SOD) is a secretory glycoprotein found mainly in the interstitial spaces, although ~1% is found in the plasma, lymph and
synovial fluids. It is the main extracellular antioxidant
enzyme in the lung. EC-SOD has a high affinity for glycosaminoglycans, such as heparan sulphate, and therefore >98% of the enzyme is found bound to heparan
sulphate in the connective tissue matrix. EC-SOD is
ideally localized to play an important role in attenuating tissue damage secondary to oxygen radicals inhaled
in cigarette smoke and generated by activated inflammatory cells.
A polymorphism in the EC-SOD gene results in the
substitution of arginine to glycine at amino acid position 213 [134, 135]. Approximately 2% of a random
population were found to be heterozygous for the substitution [135]. This mutation (R213G) is located in the
heparin-binding domain and results in a ~30 fold increase in the serum enzyme concentration, presumably due
to a failure of EC-SOD to remain bound to interstitial
glycosaminoglycans. A 10 fold increase in serum ECSOD has been reported in a lung transplant patient with
end-stage emphysema [136]. However, the R213G allele
was not present in this patient, suggesting that further
variants of this gene remain to be identified.
Immunoglobulin deficiency
Secretory leucocyte proteinase inhibitor
The role of hereditary immunoglobulin A (IgA) and
immunoglobulin G (IgG) deficiency in the aetiology of
COPD has been examined in several studies [130, 131].
Patients with IgA deficiency, either alone or in combination with IgG deficiency, are known to have recurrent
respiratory infections [132]. In a study of IgA deficient
individuals, four were found to have decreased levels
Secretory leucocyte proteinase inhibitor (SLPI) is a
12 kDa serine antiprotease found in a variety of mucous
secretions, including those of the respiratory tract. SLPI
is produced locally in the lung by airway epithelial cells
and is able to inhibit neutrophil elastase [137]. Therefore,
SLPI may play an important role in the prevention of
tissue damage by neutrophils during inflammation. ABE
Human leucocyte antigen locus
A . J . SANDFORD ET AL .
1388
et al. [138] screened 114 individuals for polymorphisms
in exons 2, 3 and 4 of the SLPI gene. The subjects included individuals with various α1-antitrypsin genotypes and 10 early onset COPD patients who did not have
α1-antitrypsin deficiency. However, no mutations were
discovered, which suggests that structural alterations in
the SLPI protein do not play a major role in the pathogenesis of COPD.
5.
6.
7.
8.
Cathepsin G
Cathepsin G is a serine protease, and mutations in the
gene for this enzyme may predispose individuals to
COPD [139]. Therefore, exons 1–5 of the cathepsin G
gene were screened for mutations in 180 individuals. A
mutation was found in exon 4, which resulted in an
amino acid substitution at position 125, but it was not
associated with COPD.
9.
10.
11.
Summary
In this manuscript, we have reviewed evidence for a
genetic component to COPD and have described the
genes that could contribute to the genetic risk. The diagnosis of COPD is based on decreased expiratory airflow, and it is possible that different pathophysiological
processes contribute to this phenotype within and between patients. For example, bronchial smooth muscle
cell hypertrophy, inflammatory narrowing of peripheral airways and loss of elastic recoil may contribute to
a different extent in certain individuals. Susceptibility
to these processes may have differing genetic bases. A
search for genes that increase susceptibility to airflow
obstruction among smokers may have implications beyond the development of COPD. In epidemiological
studies, a decrease in FEV1 has been shown to be a
marker of premature mortality from other causes [140].
It is possible that an excessive pulmonary response to
inhaled toxins and pollutants will serve as a marker of
polymorphisms that increase susceptibility to other inflammatory and degenerative diseases. The development
of rapid, inexpensive molecular methods to screen for
specific polymorphisms will allow an increased capacity
to identify risk genotypes. This has profound relevance
for the conduct of clinical investigations of environmental risk, therapeutic interventions and clinical screening.
12.
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